TWIK-related spinal cord K+ (TRESK) channel belongs to the two-pore-domain K+ (K2P) channel family and is the only K2P channel that exhibits Ca2+-dependent activation. Previous studies have shown that TRESK is a major background K+ channel in primary afferent neurons and controls neuronal excitability in both normal and disease settings. Despite the recent progress in the studies of TRESK and other K2P channels, it is still not clear whether endogenous TRESK channels are activated by intracellular Ca2+ and if so, what is the source of the Ca2+ and to what degree this contributes to the overall background K+ currents and neuronal excitability. Recently, a frameshift mutation in the KCNK18 gene encoding human TRESK subunit has been associated with migraine with aura in a large pedigree. The mutation results in the truncation of the TRESK protein. We have expressed the mutant TRESK subunit in cultured trigeminal ganglion (TG) neurons and found that it has a dominant-negative effect on the endogenous TRESK currents. Furthermore, neurons expressing mutant TRESK subunits exhibited a lower current threshold to elicit action potential as well as a higher spike frequency in response to supra-threshold stimuli, indicating that the mutation resulted in hyper-excitability of TG neurons. Interestingly, we have shown previously that a migraine-associated P/Q-type voltage-gated Ca2+ channel mutation also results in hyper-excitability of TG neurons, raising the possibility that P/Q and TRESK channels may regulate TG neuron excitability through a common pathway. The research objective of this proposal is to test the hypothesis that P/Q-type Ca2+ channels and TRESK K+ channels are functionally coupled in a subpopulation of TG nociceptive neurons to control neuronal excitability. Specifically, we hypothesize that Ca2+ influx through P/Q-type channels activates TRESK channels via the Ca2+/calmodulin-calcineurin signaling pathway, thereby regulating the excitability of TG neurons. First, we will investigate whether the endogenous TRESK channel activity is necessary for P/Q-type channels to regulate TG neuron excitability. Secondly, we will test whether P/Q-mediated Ca2+ influx is necessary for the endogenous TRESK channels to control the excitability of TG neurons. In addition, we will use HEK cells co-expressing P/Q and TRESK channels as a platform to explore the mechanisms underlying the P/Q-TRESK coupling. We will investigate whether the rise in local Ca2+ near P/Q channels is sufficient to enhance TRESK activity and whether the Ca2+ preferentially binds to the calmodulin pre-associated with P/Q-type channels. We will also test whether the P/Q-TRESK coupling requires the activation of calcineurin anchored to the TRESK channels. Lastly, we will investigate whether P/Q and TRESK channels interact with each other within a protein complex. The outcome of this study will lead to better understanding of the mechanisms underlying the P/Q-TRESK as well as its functional significance. Ultimately this will shed light on the common mechanisms through which P/Q and TRESK channel mutations cause migraine headache.